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UNITED STATES DEPARTMENT OF AGRICULTURE 

BULLETIN No. 340 

Contribution from the Bureau of Animal Industry 
A. D. MELVIN, Chief 




.^?9^'^^U 




J'^^'^U 



Washington, D. C, 



PROFESSIONAL PAPER 



December 27, 1915 



EXPERIMENTS IN VACCINATION AGAINST 
ANTHRAX.^ 

By Adolph Eichhorn, Chief of the Pathological Division. 



CONTENTS. 



Prevalence of anthrax and methods of con- 
trol 1 

Protective vaccination 2 

Production of serum 4 

Standardization of the serum 7 

Experimental data 8 

Hyperimmunization of horses 8 

Serum tests 9 



Page. 

Preparation o f spore vaccine 10 

Technic of administration 11 

Test of the simultaneous method on cattle 

and sheep 13 

Field tests 13 

Treatment of anthrax in man 15 

Concentration of serum 15 

Conclusions 16 



PREVALENCE OF ANTHRAX AND METHODS OF CONTROL. 

Anthrax is a disease that is widely spread throughout the world, 
and in the United States it is being recognized as one of the most 
destructive scourges of live stock. In certain sections it is more 
prevalent than in others, particularly in the Southern States, and 
since no determined effort has been made toward its suppression it 
appears to be on the increase, its presence now being recorded in 
localities where it has never before been recognized. 

As the spores of the causative agent of anthrax retain their 
virulence and remain lodged in the soil in an active state for many 
years in the infected localities, it is very difficult to prevent the 
spread of the infection, and the eradication of the disease is thereby 
rendered a most serious problem. 

Various factors have to be considered in the prophylactic control 
of anthrax, such as the prevention of the continued impregnation of 

1 The author was ably assisted in the technical part of this work by Dr. Raymond A. Kelser, assistant 
in pathology and bacteriology, to whom credit is due for the painstaking and careful execution of the 
details of the experiments. 

Note. — This bulletin is of interest to stock raisers and veterinarians generally. 



14044°— 15- 



Monograph 



C.l\ria^ 



Z. 



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V 

2 BULLETIN 340^, U. S. DEPARTMENT OF AGRICULTURE. -" b 

the soil with the virus by the proper disposition of the carcasses of 
animals that have died of the disease, the destruction of the virus 
contained in the soil by its proper drainage and cultivation, and the 
prevention of outbreaks through the immunization of the susceptible 
animals. 

In order to attain the greatest success in the control and eradica- 
tion of the disease, it would appear that the best results can be ac- 
complished only through proper attention to all of the above factors. 
The execution of these measures would require the earnest coopera- 
tion of the stock owners, but even then, on account of the peculiar 
geographical conditions of certain parts of the country, the drainage 
and cultivation of the land would not always be feasible, and our 
efforts must therefore be directed principally toward the sanitary 
measures and protective vaccination. The enforcement of proper 
sanitary police regulations in connection with the control of anthrax 
would no doubt effect a material reduction of the disease, but un- 
fortunately it is rather a difficult task to obtain the cooperation of 
the interested parties. 

The proper disposition of the infective material, particularly the 
carcasses, should be considered of the utmost importance, since such 
material constitutes the greatest source of danger toward the spread- 
ing of the disease. Drainage from the soil polluted by infected 
carcasses may carry the infection to distant points and deposit the 
spores over large areas hitherto uninfected. Buzzards and other birds 
(Dalrymple), dogs, and eyen flies may also carry the infection from 
such sources into uninfected localities. Therefore, in an effort to 
control the disease, an educational propaganda must be carried out 
and stringent compulsory measures adopted for the proper disposi- 
tion of the infective material from premises where the disease appears 
among the stock. 

PROTECTIVE VACCINATION. 

A material reduction and a checking of the disease may be suc- 
cessfully accomplished by periodical vaccination of all stock in in- 
fected localities. This method, even if practiced alone, would have 
splendid results in minimizing the losses from the disease in anthrax 
localities. However, such vaccination must be carried out regu- 
larly and irrespective of whether the disease has already appeared 
on the premises. 

Fortunately we have at our command various methods of vacci- 
nation which have proved highly efficient in the production of im- 
munity from anthrax. As a matter of fact, this was one of the first 
infectious diseases in which protective vaccination was successfully 
demonstrated, and we are indebted to Pasteur for devising the pro- 

D. Of.^ 0. 
JAW 6-'*l916 



EXPEEIMENTS IN VACCINATION AGAINST ANTHEAX. 3 

cedure of the vaccination for this purpose. Pasteur proved that 
anthrax bacilli when cultivated at a temperature of from 42° to 43° 
C. will gradually lose their virulence, and also that when removed 
from such an attenuating temperature and cultivated under normal 
incubation temperature they will not change their pathogenicity. 
Thus cultures attenuated for 24 days will be pathogenic for mice 
but not for guinea pigs and rabbits, whereas if attenuated for only 
12 days at the higher temperature they will be virulent for mice 
and guinea pigs but not for large rabbits. The attenuated cultures 
will retain their reduced virulence under ordinary conditions, and 
only in very exceptional instances has any increase of virulence 
been observed. This characteristic of the anthrax bacillus led Pas- 
teur to employ the attenuated forms of the anthrax cultures for 
vaccination purposes. Accordingly he prepared a weakened vac- 
cine from cultures which had been attenuated for 24 days {premier 
vaccin), and for a second injection cultures which had been attenu- 
ated for 12 days {deuxietne vacciji}. In the epoch-making demon- 
stration at Pouilly le Fort, before a commission appointed by the 
French Government, he successfully demonstrated its effectiveness 
on sheep and cattle. In this^instance the vaccinated animals with- 
stood the injection of virulent anthrax bacilli, whereas the controls 
died. Since that time vaccination against anthrax by the Pasteur 
method has been very extensively employed throughout the world. 
Many millions of animals have been vaccinated by this method, and 
the results in general must be considered very favorable. 

At the same time it must be aclmowledged that in vaccination by 
the Pasteur method it is essential to have a potent vaccine and one 
wdiicli is properly tested for its pathogenicity. There are disad- 
vantages in this method of vaccination and these must be given due 
consideration. The unstable keeping quality of the Pasteur vaccine 
is a very important factor to be considered. Experience in this line 
has proved that Pasteur vaccine may deteriorate within a very short 
time after its preparation, and this has also been demonstrated during 
the work of the Bureau of Animal Industry in the control of the 
manufacture of biological products, when periodical tests were 
undertaken with those of various manufacturers. In repeated in- 
stances a vaccine proved inert within three months of its preparation. 
At other times it remained potent for a period of a year. This no 
doubt is due to the method of preserving and handling the product. 
AVhen exposed to light and warm temperature it deteriorates very 
rapdily, and when it is considered that the products of manufacturers 
may be stored under unfavorable conditions in branch houses and on 
the shelves in rural drug stores the loss of potency can be readily 
explained. For this reason it seems wise to reduce the time limit for 



4 BULLETIN 340, U. S. DEPARTMENT OF AGRICULTURE. 

the use of Pasteur anthrax vaccine to three months from the date of 
its preparation. 

The injection of an inert product into animals would impart to 
the stock owners and veterinarians who employ it a false sense of 
security and would bring this method of vaccination into disrepute. 
At times no doubt great losses have resulted from the application 
of inert vaccines. 

Other disadvantages of the Pasteur method which must be con- 
sidered are, first, that it requires two handlings of the animals 
before immunity is established; second, that the losses from vac- 
cinations are not insignificant; third, that its standardization is 
not carried out very accurately; and, fourth, that its administration 
in herds where the disease has already made its appearance is liable 
to induce the disease, through the reduction of the resistance of the 
animal during the process of vaccination, and for this last reason it 
is best adapted for use only with herds in which the disease has not 
yet appeared. 

These deficiencies of the method have been recognized by many 
investigators, who have endeavored to devise other methods of 
vaccination, and particular attention has been directed toward the 
preparation of a spore vaccine, because of its superior keeping 
qualities. In Russia at the present time the method of Zenkowsky, 
and in Hungary a spore vaccine prepared by Detre, are being suc- 
cessfully employed; although, aside from their keeping qualities, 
these products have all the other disadvantages of the Pasteur 
method. Successful vaccination by spore vaccines was also demon- 
strated by Nitta, in Japan, and by others. Other means of vaccina- 
tion with attenuated living cultures, aggressions, dead bacteria, etc., 
were tried, but proved of no advantage. 

Sclavo, Sobernheim, and others have established that injections 
of increasing amounts of virulent cultures into immune animals 
produced a serum which has great protective value against anthrax. 
Such protective serum may be produced in the various susceptible 
animals. 

PRODUCTION OF SERUM. 

The animals which are selected for the preparation of serum are 
subjected to a preliminary treatment either by sero- vaccination or by 
Pasteur's method, then at certain regular intervals they are infected 
with increasing doses of virulent anthrax cultures. For this purpose 
they receive in about 10 to 14 days following the preliminary treat- 
ment an injection of from 0.005 to 0.001 of a loopful of virulent cul- 
ture. In sheep it is advisable to exercise greater care, especially at 



EXPERIMENTS IN VACCINATION AGAINST ANTHRAX. 5 

the first injection of virulent material, when a very small quantity 
of culture should be employed, whereas in cattle and horses it is not 
necessary to employ less than 0.005 of a loopful. The first injection 
of virulent culture is usually followed by a considerable reaction, 
inasmuch as the animals usually develop a febrile condition which 
persists for several days. The subsequent inoculations are then car- 
ried out at intervals of from 2 to 3 weeks in such a way that the 
dose is soon increased to a loopful, then to several loopfuls, and 
gradually to several agar cultures, and, finally, to an injection con- 
sisting of several large mass cultures. This is quite easily accom- 
plished in cattle and horses, and in 3 to 4 months the animals 
may become so tolerant to this injection that they will withstand 
the subcutaneous inoculations of two to three mass cultures without 
noteworthy reaction. At times considerable extensive local infiltra- 
tion may follow the injection, which, however, retrogresses within 
a short time and the general condition of the animals is only slightly 
influenced. In sheep the immunization causes greater difficulties 
on account of a greater susceptibility of these animals, and it is 
difficult to prevent a very small percentage of the animals which are 
being used for serum production from dying in the course of the hyper- 
immunization. Nevertheless it is possible, even in sheep, to produce 
such an immunity that they will withstand the injection of several 
mass cultures without reacting.^ 

The more virulent the strain of the anthrax culture which has 
been used for the treatment of the animals the more care must be 
exercised in the course of the hyperimmunization, but in that case 
the anthrax serum would also be more potent. Therefore, it is advis- 
able to use anthrax strains which have been recently obtained from 
fatal infections. It is also advisable to use strains of different 
origin for the immunization. It is immaterial whether bouillon 
cultures are used or suspensions from agar cultures, but it is more 
practical to use the latter method for the inoculating material, since 
in this instance the quantity of fluid to be injected may be limited 
to a relatively small amount. Quantities of 500 to 1,000 c. c. of 
the bouillon cultures cause, as can be readily seen, considerable tech- 
nical difficulty for injection, whereas the suspensions from four or 
five mass cultures may be readily distributed in 50 to 60 c. c. of 
-fluid. Fresh cultures which have been cultivated for about 24 hours 
at 37° C. are as a rule more suitable for inoculation, whereas older 
cultures with pronounced spore formations possess no advantages 
over the young cultures. 

' Bureau of Animal Industry Bulletin 137, "Anthrax, with special reference to the pro- 
duction of immunity," by Charles F. Dawson. 1911. See p. 43. 



6 BULLETIN 340^ U. S. DEPARTMENT OF AGRICULTURE. 

The inoculations should be made subcutaneously. Intravenous 
injections as first employed by Sclavo are less effective. The potency 
of the anthrax serum is in no way increased by this method of 
immunization. Besides there exists the danger of emboli when in the 
later stages of the immunization process larger amounts of culture 
material have to be administered. Animals which have been treated 
with subcutaneous injections wdll produce finally an anthrax serum 
of remarkably high potency. 

As a rule the animals which have received one to two agar cultures 
show a specific protective action of their serum, but for practical 
purposes it is not advisable to use such a serum. Generally only 
when the animals stand one-half to one mass culture is the potency 
of the serum sufficiently strong. A similar condition is manifested 
in animals used for the production of immune serums for other 
diseases, the individuals showing a varying response to the injection 
for the production of immune bodies, i. e., an animal will at times 
produce a potent serum relatively early, whereas another with the 
same method of treatment wdll develop a serum of the same potency 
only after a considerably longer preparatory treatment. Accord- 
ingly, from observation it has been noted that sheep produce the 
most potent serum, and in this species of animals the individual 
differences are of almost no consequence, so that almost every animal 
produces a good anthrax serum. Horses also produce a potent serum, 
although single individuals may show great variations. The anthrax 
serum from cattle is quite potent, but in its protective value it does 
not equal horse and sheep serum. 

It is best to draw the blood 14 to 16 days after the last injection; 
an earlier bleeding should be avoided. Not infrequently it occurs 
that animals after an apparent recovery following the inoculation 
reaction and after a period in which they are free of fever on the 
eighth or ninth day suddenly develop a rise in temperature. This 
has been established by Sclavo and Burow. Then, again, repeated 
regular blood examinations showed that at this time and even later, 
up to the tenth and eleventh days following inoculation, occasional 
anthrax bacilli may appear in the blood of the animals in greater 
numbers. 

The bleeding is carried out in the ordinary way, and the blood is 
collected in large sterilized glass cylinders or similar receptacles of 
about 2 or 3 liters capacity. Seven or eight liters of blood may be 
drawn from cattle, about the same quantity from horses, and about 
1 to H liters from sheep. After 2 or 3 days another bleeding is 
made. In this instance, however, only a small quantity of blood 
should be drawn. The animals resist these operations very readily, 
and after a lapse of 14 days they are ready for another injection, 



EXPEEIMENTS IN VACCINATION AGAINST ANTHRAX. 7 

which is then followed in from 14 to IG days by repeated bleedings. 
Thus, in the period of a year, the same animals may be bled 10 to 
11 times, and such animals can be used in this way for several years, 
alternating the injections with the bleedings, provided they are kept 
in a well-nourished and healthy condition. 

In order to obtain the largest possible yield of serum from the 
blood drawn into the glass cylinders a weight is attached to the same 
and released onto the clotted blood in about 12 hours after being 
drawn. The diameter of the weight is about half an inch less than 
the cylinder and its weight is about 2 pounds. In about 24 hours 
the clear serum is then siphoned into sterile bottles and preserved 
with 0.5 per cent of carbolic acid. If proper precautions have been 
practised, it is not necessary to pass the serum through Berkefeld 
filters ; however, if there is the slightest doubt as to its sterility, it is 
desirable to filter the serum before bottling. It is advisable to dis- 
tribute the serum in various-sized brown bottles, which should be 
securely corked and paraffined. 

STANDARDIZATION OF THE SERUM. 

The testing of the serum must be carried out primarily to determine 
its potency. It is to be regretted that for this purpose there are no 
accurate or definite methods known, and it is almost impossible to 
establish the absolute protective value of the serum, because the 
animals on which it is being tested are so very highly susceptible to 
the disease. Nevertheless, it is possible to establish a relative value 
for all practical purposes through laboratory experiments, and some 
investigators believe that rabbits are best adapted for the purpose. 
The standardization test as recommended by Sobernheim is still em- 
ployed by various investigators. This test is carried out as follows : 



Potency test for anthrax serum (Sobernheim). 



Rabbit. 



First injection. 



Second injection. 



A.. 

B 

C 

D 

E 

F Ccontiol). 



G (control). 



2 c. c. of immune serum (intravenous) 

3 c. c. of immune serum (intravenous) 

4 c. c. of immune serum (intravenous) 

5 c. c. of immune serum (intravenous) 

6 c. c. of immune serum (intravenous) 

0.(X)1 loopful of a suspension of virulent 

anthrax bacilli in 1 c. c. of 0.7 per cent 
sodium-chlorid solution. 
do 



Follow immediately by a subcutaneous 
injection of 0.001 loopful of a suspension 
of virulent anthrax bacilli in 1 c. c. of 0.7 
per cent sodium-chlorid solution. 



According to extensive experience, a serum is considered potent 
and satisfactory for immunization purposes when at least two of 
the five rabbits given the serum remain alive and the others die 
later than the control animals. Should more than the two animals 
remain alive, while the control animals die in about 48 hours, the 



8 ' BULLETIN 340, U. S. DEPARTMENT OF AGRICULTURE. 

serum has an extraordinary potency. It should be noted that it 
does not follow that those rabbits which receive the smallest serum 
doses should die, since not infrequently they may remain alive when 
the rabbits receiving larger doses succumb. 

This method of standardization has not been proved as accurate 
and reliable as the test recommended by Ascoli, and which has been 
employed in the experimental work with serum prepared in con- 
nection with our experiments. In this test a 24-hour-old attenuated 
bouillon culture is used, which is of such virulence that when intro- 
duced subcutaneously in a 0.25 c. c. dose into 350-gram guinea pigs 
it will kill them in from two to three days. These test cultures must 
be previously standardized in such a way that they will kill guinea 
pigs which 24 hours previously have been injected intraperitoneally 
with 2 c. c. of normal serum. Guinea pigs treated in the same man- 
ner and M'ith the same dose of titrated standardized immune blood 
serum must remain alive. 

The testing of the serum is carried out on six guinea pigs, each 
receiving intraperitoneally 2 c, c. of the serum to be tested, and 24 
hours later the established dose of the test culture is injected sub- 
cutaneously in the axillary region. The serum is considered satis- 
factory for immunization purposes if at least four of the guinea pigs 
remain alive over six days while the control animals die within 
three or four days. For protective and curative purposes in man, 
only such serum should be selected which, by carrying out the same 
conditions of the test, protect the guinea pig in 0.5 to 1 c. c. doses. 

EXPERIMENTAL DATA. 

HYPERIMMUNIZATION OF HORSES. 

On September 8, 1914, two horses, Nos. 48 and 96, were vaccinated 
against anthrax according to Pasteur's method. On September 29 
these two horses were given approximately 0.01 of a loopful of 
virulent anthrax bacilli subcutaneously. Horse No. 48 showed no 
apparent reaction following the injection. Horse No. 96, however, 
developed local anthrax at the point of inoculation. The swelling 
became enlarged and there was a considerable area of edema below 
the same. This condition persisted for approximately a week, and 
finally disappeared. The animal, however, showed no appreciable 
rise in temperature during this period. 

The following table gives in detail the process of hyperimmuni- 
zation : 



EXPEEIMENTS IN VACCHSTATION AGAINST ANTHRAX. 
Hyperimmimization of horses Nos. J/S and 96, 



Date. 



Amount of virus given each horse. 



Result. 



1914. 
Sept. 29.... 



0.01 loopful. 



Oct. 24.. 
Nov. 15. 
Dec. 9... 



Dec. 29. 
Jan. 19. 
Feb. 6.. 



Mar. 5. 



Mar. 31. 



Apr. 19. 
Apr. 28. 
May 11.. 
May 24.. 
June 12. 



1 loopful 

10 loopfuls 

5 c. c. of an emulsion, representing one- 
half growth of agar culture. 



20 c. c. of emulsion, representing washing 

of growth from 2 agar cultures. 
30 c. c. of emulsion, growth from 8 agar 

cultures. 
40 c. c. of emulsion, growth from 2 mass 

cultures from flasks, surface area 6 by 

2J inches. 

50 c. c. of emulsion, growth from 4 mass 

cultures from flasks, surface area 6 by 

2% inches. 
50 c. c. of emulsion, growth from 8 mass 

cultures from flasks, surface area 6 by 

2i inches. 

....:do 

do 

do 

do 

do 



No apparent reaction ia horse 48. Horse 
96 developed anthrax at point of inocula- 
tion; large swelling; edema of neighbor- 
ing tissue. Persisted about one week. 

No noticeable reaction in either animal. 
Do. 

Horse 48 showed a temperature of 102.2° 
the following day; horse 96, 101°. Both 
animals developed a small, hard nodule 
at point of inoculation. 

Both animals developed small abscess at 
point of inoculation. 

No reaction. 

Slight reaction in horse 96. Horse 48 
showed quite an intensive reaction , devel- 
oping a large swelling at point of inocu- 
lation; persisted several days. 

No apparent reaction. 



Slight local reaction in each case. 



Do. 
Slight rise in temperature in both cases. 
Slight temperature and local reaction. 

Do. 
Slight local reaction. 



In the above work four strains of anthrax bacilli were used, known 
to us as "Davis," "6071," "Burt," and " Boener "— the first two 
strains being highly virulent types and the latter two very much 
weaker. In all cases where the larger amounts of the virus were 
given the injections were made at 4 to 6 different points in order to 
minimize abscess formation. 

It might be well also to state here that the irregularity in the time 
between injections was due to the fact that this work was interfered 
with by the outbreak of foot-and-mouth disease in this country, and 
for this reason it was also impossible to subject the blood to periodical 
tests to ascertain its immunizing value at the different intervals 
between injections. Experience proved that horses may produce 
highly potent serum following the injection of the first or second 
mass cultures. It is therefore advisable to subject the blood of the 
animals to periodical tests for potency throughout the course of 
immunization. 

On June 25, 1915, 6 liters of blood were drawn from each horse 
into the glass bleeding cylinders previously described. Since this 
date these animals have been bled regularly, 6 liters being taken 
from each horse, and an injection of virus made in the intervals 
between bleedings. 

SERUM TESTS. 

In standardizing our serum, that taken from each horse was tested 
separately. The following procedure was carried out: Three series 



10 



BULLETIlSr 340, U. S. DEPAETMENT OF AGRICULTURE. 



of guinea pigs were inoculated intraperitoneally with varying 
amounts of serum, and 48 hours later were injected with 0.25 c. c. 
of a 24-hour bouillon subculture of an attenuated strain Iniown as 
" Davis D." This culture had been attenuated by growing it at a 
temperature of 42°-43° C. for a period of 20 days. Previous tests 
of this culture showed that it was uniformly pathogenic for guinea 
pigs, killing them in two to three days, but it failed to kill rabbits. 
The results of this test are contained in the following table : 

Standardization tests of anthrax serum {serum injected intraperitoneally; 
virus 24 hours later subcutaneously) . 

SERUM 48. 



Guinea pig No. 


Amount of 
seram. 


Amount of 
viius. 


Result. 


1 


1.0c. c. 
1.5C.C. 
2.0 c. 0. 
2.5 c. c. 
3.0 c. c. 
3.5C.C. 


0. 25 c. c. 
0.25 c. c. 
0. 25 c. c. 
0.25 c. c. 
0. 25 c. c. 
0.25 c c. 


Remained alive. 


2.. 


Died on third day. 
Remained alive. 


3 


4. 


Do. 


5 


Do. 


6 


Do. 







SERUM 96. 



1 


1.0c. c. 
1.5C.C. 
2.0C.C. 
2.5 c. c. 
3.0 c. C. 
3.5 c. c. 


0. 25 c. C. 
0. 25 c. c. 
0. 25 c. c. 
0. 25 c. c. 
0. 25 c. c. 
0.25 c. c. 


Remained alive. 


2. 


Do. 


3 


Died on third day. 


4 


Remained alive. 


5 


Do. 


6 


Do. 







NORMAL HORSE SERUM. 



1... 


1.0 c. c. 
1.5C.C. 
2.0 c. c. 
2.5c. c. 
3.0 c. c. 
3.5C.C. 


0. 25C.C. 
0. 25 c. c. 
0.25C.C. 
0.25e.c. 
0. 25C.C. 
0.25C.C. 


Died on fourth day. 


2 


Died on third day. 


3. 


Died on fourth day. 


4 


Remained alive. 


6 


Died on lourth day. 


6... 


Died on third day. 







In view of these results it was decided to use the " Davis D " cul- 
ture in the preparation of our spore vaccine, to be used simultane- 
ously with the serum. 

Extensive tests to determine whether or not the immune serums 
possessed a bactericidal property proved negative. 

PREPARATION OF SPORE VACCINE. 



The four cultures used for the hyperimmunization of the horses 
were attenuated at a temperature of 42.5° C. for varying periods. 
From time to time they were tested for their pathogenicity by inocu- 
lation into mice, guinea pigs, and rabbits. The cultures, which were 
removed from the incubator after 20 days of attenuation, proved sat- 
isfactory for the purpose, inasmuch as the test inoculation demon- 



EXPEEIMENTS IN VACCINATION AGAINST ANTHEAX. 11 

strated their virulence for the mice and guinea pigs, but not for 
rabbits. 

P'or the purpose of producing a spore vaccine it is desirable to use 
a peptone-free agar media and after inoculation with the attenuated 
culture to grow the organism at a temperature of 37.5° C. for 4 to 7 
days, by which time an abundance of spores will have formed. The 
growth is then washed from the slants and collected in a sterile 
flask and heated at a temperature of 60° C. for one-half hour, to 
destroy the vegetative forms of the organism. A measured quantity 
of this suspension can then be plated out in the usual manner and the 
spore content of 1 c. c. of the suspension established. A dilution can 
then be made to the desired amount for inoculation purposes. Thus, 
if it is desired to use for vaccination 1,000,000 spores, it is best to 
dilute the vaccine to a quantity of which 1 c. c. would contain this 
number. Of such vaccine 1 c. c. would constitute the dose for cattle 
and horses, with correspondingly smaller doses for calves and sheep. 

In all forms of vaccination against anthrax in sheep the greatest 
care must be exercised, since these animals are very susceptible to the 
disease, and at times vaccines which have no ill effects on cattle will 
prove fatal to sheep ; therefore the dose of the spore vaccine for sheep 
should not be more than one-fourth the amount given cattle. 

In the preparation of spore vaccines it is essential to submit every 
lot to a test for pathogenicity by inoculating approximately 250,000 
spores — that is, 0.25 c. c. of the standard suspension — into guinea 
pigs and rabbits before employing the same for vaccination purposes. 
The guinea pigs should die in from 2 to 5 days, whereas the rabbits 
should remain alive. 

In consideration of the keeping qualities of the spore vaccine, large 
lots can be prepared without fear of deterioration. In the bottling 
and storing of the same, however, proper care should be taken to 
prevent contamination. 

TECHNIC OF ADMINISTRATION. 

For immunization purposes by the simultaneous method the serum 
should be injected first. It is desirable to divide the herd into groups 
of 10 or 12 and inject first each animal of the group with the serum, 
following this with the injection of the spore vaccine. The serum 
should be injected on one side, either on the neck or back of the 
shoulder, and the spore vaccine on the other side, the injections being 
made subcutaneously. 

In herds where the disease has already madje its appearance it is 
necessary to take the temperatures of all the animals and to subject 
to the simultaneous vaccination only those that show no rise in tem- 



12 BULLETIN 340, U. S. DEPARTMENT OF AGEICULTTJEE. 

perature. All others should be given the serum- alone treatment in 
doses varying in accordance with the severity of the symptoms mani- 
fested by the individual animals. If the examination reveals a con- 
siderable number of infections, it is advisable to use the serum alone 
for all the animals, and in 3 or 4 weeks to revaccinate by the simul- 
taneous method. 

The dosage should depend on the potency of the serum, serum of a 
high potency naturally being most desirable ; thus, in some instances 
serum in .5 c. c. doses for large animals and 3 c. c. for smaller ones was 
found to be effective for immunization purposes. Unfortunately all 
hyperimmune animals do not yield serum of such high potency, and 
for this reason it is obvious that accurate potency tests should be 
carried out by the producer of the serum. 

In the treatment of anthrax, serum should be administered in large 
doses. An animal showing only a high temperature, with no other 
manifestations of the disease, should be given from 30 to 50 c. c, but 
if the gravity of the disease is pronounced, 100 c. c. should be ad- 
ministered. In ahnost every instance a drop in temperature may be 
observed and a diminishing of the severity of the symptoms. At 
times, however, a relapse occurs about the second or third day fol- 
lowing the serum injection, when it becomes necessary to administer 
another dose of serum. It has been proved that animals affected 
with anthrax, even after the bacilli are found in the blood circulation, 
may recover after an injection of potent serum. 

The simultaneous treatment, as in the Pasteur treatment, may at 
times result in a temperature and systemic reaction in the animals. 
These manifestations are indicated by an elevation of temperature 
and sometimes by a swelling at the point of inoculation of the spore 
vaccine. These sjnnptoms, however, are usually of short duration, 
and only in very exceptional cases will they result in the loss of the 
animal. However, if the reaction following the injection of the spore 
vaccine threatens the life of the animal, a second injection of serum 
should be administered. 

The anthrax serum injected simultaneously with the vaccine has a 
counteracting effect upon the reaction which may follow the injec- 
tion of the spore vaccine during the process of immunization. 

At times anaphylactic reactions are observed as a result of the 
serum injected, especially in cases where the serum is foreign to the 
animals treated. These manifestations appear as a rule within one- 
half hour after injection, in the form of urticarialike eruptions, 
swelling of the head, slight chills, and rise in temperature. More 
severe symptoms have also been noted to follow such injections, but 
they ahnost invariably subside within a few hours. 



EXPEEIMENTS IN VACCINATION AGAINST ANTHRAX. 13 

TEST OF THE SIMULTANEOUS METHOD ON CATTLE AND SHEEP. 

A series of experiments was conducted at the experiment station of 
the Bureau of Animal Industry at Bethesda, Md., to establish the 
efficiency of the simultaneous method of anthrax immunization on 
cattle and sheep. 

For this purpose 6 head of cattle and 5 sheep were given the simul- 
taneous injection of anthrax serum and spore vaccine. Three weeks 
subsequent to immunization they were subjected to infection tests 
which consisted of a subcutaneous administration of 0.25 c. c. for the 
cattle and 0.125 c. c. for the sheep of blood from a guinea pig which 
had died from an artificial infection with our most virulent strain of 
anthrax. 

The microscopic examination of the blood of the guinea pig showed 
it to be heavily charged with anthrax bacilli, but in order to make 
the test as severe as possible it was deemed advisable to use such exces- 
sive amounts. Three additional cattle and two sheep were used as 
checks, receiving only the virulent blood. As a result of this infec- 
tion all animals manifested an elevation of temperature ranging from 
103° to 107° F. The control animals especially Avere markedly 
affected with typical manifestations of anthrax and all succumbed 
within two to eight days following infection. All but one of the 
vaccinated sheep succumbed to anthrax, but at a later date than the 
check animals. Of the immunized cattle a marked temperature reac- 
tion was noted, but all of these animals recovered with the exception 
of a small, undersized, weak calf, which died in six days following 
infection. 

While in the above test the sheep succumbed and one of the small 
calves died of anthrax, nevertheless the potency of the serum was 
demonstrated. The excessive virulent blood used for the infection 
was extraordinary and could not be compared with the amount of 
virus taken by a susceptible animal in cases of natural infection. 

FIELD TESTS. 

On June 21, 1915, Dr. E. R. Ashworth, a dairy inspector for the 
District of Columbia, notified our office that a number of deaths 
among hogs were occurring on a farm in Maryland, just outside of 
the District. The symptoms described by Dr. Ashworth pointed sus- 
piciously to anthrax. A visit was made to the farm the same morn- 
ing, and after an autopsj'^ on several animals, followed by a bac- 
teriological examination, a definite diagnosis of anthrax was estab- 
lished. This was later conclusively verified by animal inoculation 
tests. 

At that time 7 shoats and 4 sows had died of the disease and 3 shoats, 
4 sows, and 1 boar were showing symptoms of anthrax, several of the 



14 BULLETIN 340^ U. S. DEPARTMENT OF AGRICULTURE. 

sick animals manifesting the characteristic edema of the throat 
region. It is desired to make particidar mention of the boar, a fine 
pure-bred animal, which was in an almost comatose condition, show- 
ing a profuse bloody diarrhea, and a temperature of 106° F. One of 
the sows was also in a very critical condition. 

On the afternoon of June 21 the affected animals were given injec- 
tions of the immune serum, the boar receiving 100 c. c, the sows 50 
c. c, and the shoats 30 c. c. On the following day a visit was made 
to the farm to immunize the remaining hogs, which as yet had shown 
no symptoms of the disease. A. total of 138 were given protective 
doses of the serum, the larger hogs weighing 75 pounds or over receiv- 
ing 10 c. c. and the smaller animals 5 c. c. Marked improvement was 
noted in the sick animals that had been treated the day before. 

On June 23 another visit was made to the farm. All of the sick 
animals showed still further improvement. The boar was given 60 
c. c. more of immune serum and the sow that had been the most sick 
Avas given an additional 30 c. c. 

The result of this work was that every affected animal recovered, 
and up to the present time not a single death from anthrax has been 
repoi'ted in those animals that received portective doses of the serum. 

In the early part of July an outbreak of anthrax was reported 
from Queen Anne County. Md. On July 13 two inspectors from 
the bureau were detailed to make an investigation, with a view to 
using our immune serum and spore vaccine in an effort to control 
the outbreak. The disease had made its first appearance about a 
month previous to this time, when a farmer lost a cow from anthrax. 
A few days later a neighbor on an adjoining farm lost a hog from the 
disease. Following this, the disease made its appearance on five 
other farms in the immediate vicinity, the greater percentage of 
animals stricken dying of the apoplectic form of the malady. Ani- 
mals on some of the farms had been treated with single injections 
of a commercial vaccine before the arrival of our inspectors. Immu- 
nization tests were at once started with the bureau serum and spore 
vaccine, with the following results : 

The animals on six farms where losses had occurred from anthrax 
were vaccinated, the cattle, horses, and mules receiving 10 c. c. each 
of serum and 1 c. c. of spore vaccine, except, however, in cases where 
there was reason to believe an animal might be in the incul)ative 
stage of the disease, Avhen the vaccine was omitted and the dose of 
serum increased. Sheep and hogs on the infected farms were given 
the serum-alone treatment, receiving from 5 to 10 c. c. each. 

On the day subsequent to vaccination a mule on one of the farms 
showed symptoms of anthrax, there being an elevation of tempera- 
ture and a characteristic swelling on one side of the neck, the side 



EXPEKIMENTS IN VACCINATION AGAINST ANTHRAX. 15 

opposite to where the vaccine had been injected. This animal was 
given an injection of 60 c. c. of serum and made a speedy recovery. 

In ajl, 399 animals, including horses, mules, cattle, sheep, and hogs 
on farms where the disease had broken out, were treated with the 
bureau serum and vaccine. Previous to this an aggregate of 10 
cattle, 3 mules, and 13 hogs had died of anthrax on these farms. 
On the morning of the day following vaccination a cow on one of the 
farms died of anthrax. Exclusive of the above, no losses from 
anthrax have occurred on any of these farms. 

Approximately 140 animals on several other infected farms were 
vaccinated with a commercial vaccine by a representative of the 
State live stock sanitary board. Within a day or two following 
this vaccination it was reported 3 cows and 1 mule died of anthrax, 
and since then 2 more cows have died of the disease. 

Another opportunity was afforded us to test the serum and vaccine 
in an outbreak of anthrax in Noxubee County, Miss., where a num- 
ber of farms were reported to be infected with the disease. A 
quantity of serum and spore vaccine was furnished, and an inspector 
detailed from the bureau station at Birmingham, Ala., to conduct 
the work. On various farms where the disease had made its appear- 
ance a total of 125 cattle were given the simultaneous treatment. In 
addition 3 animals which showed symptoms of the disease were 
given 30 c. c. of serum alone. No deaths from anthrax occurred 
immediately following or since the vaccination, the affected animals 
having all recovered from the disease. 

USE OF SERUM IN TREATMENT OF ANTHRAX IN MAN. 

Extensive data are available on the effectiveness of anthrax serum 
for the treatment of the disease in man. It is recommended that 
from 30 to 40 c. c. of serum be injected in three or four different 
places. Should no impr-ovement follow in 24 hours an additional 
injection of 20 to 30 c. c. of serum should be administered. 

In most instances the results are very favorable, and this treat- 
ment is acknowledged to be superior to any other mode of treatment 
known for this disease. 

CONCENTRATION OF SERUM. 

Experiments are now being conducted in drying immune serum 
with a view to preparing the same in pellet form. For this purpose 
the serum has been dried in shallow pans in a serum-drying appa- 
ratus. After thorough drying it is scraped from the pans, milled 
into a fine powder, and prepared in a pellet machine into proper- 
sized pellets. The spore vaccine is also being prepared in a similar 



16 BULLETIN 340, U. S. DEPAETMENT OF AGRICULTURE. 

manner. This procedure would greatly simplify the administration 
of the serum and vaccine and, besides, the products would be in a 
form least likely to deteriorate or become contaminated. 

The proteids containing the protective bodies of the serum have 
also been successfully precipitated through fractional saturation of 
the serum with ammonium sulphate, and further work along this 
line is now being conducted. However, this work and the work on 
the drying and concentration of the products are still in the experi- 
mental stage, and it is our aim to properly work out a method most 
suitable for immunization of animals in the field. 

CONCLUSION. 

1. Horses are suitable for the production of highly potent anthrax 
serum. Serum of such horses should protect large animals in 10 c. c. 
doses. 

2. The use of the serum-alone treatment is indicated in cases where 
the infection has already occurred in a herd. Since the serum con- 
fers only a passive immunity, it is advisable to revaccinate the herd 
in from three to five weeks by the simultaneous method. 

3. The serum possesses great curative value. Depending on the 
severity of the infection, the curative dose is from 30 to 100 c. c. ; the 
injection to be repeated if necessary. 

4. For the simultaneous treatment a spore vaccine, carefully stand- 
ardized, is preferable to the ordinary Pasteur vaccine. 

5. Spore vaccine should be employed also in preference to the 
Pasteur vaccines for immunization with vaccine alone. This vaccine 
has a decided advantage over the Pasteur, because of the possibility 
of more accurate dosing and because of its better keeping qualities. 

6. Experiments with concentrated serum and dry spore vaccine 
are very promising. This method would greatly simplify the vac- 
cination process and also insure the product against subsequent con- 
tamination and deterioration. 



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